In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes.

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.

Should we be afraid of long-term cardiac consequences in children with multisystem inflammatory syndrome? Experience from subsequent waves of COVID-19.

The purpose of the study was to assess and compare short- and long-term cardiac complications of the multisystem inflammatory syndrome in children (MIS-C) by predominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants throughout the pandemic. The analysis of prospectively collected data comparing cardiac complications of MIS-C during and after hospitalization across the original/alpha, delta, and omicron waves. Cardiac complications were defined as cardiac failure with systolic function impairment or hypotension or abnormalities in echocardiographic findings (decrease in LVEF, FS, valvular insufficiency, pericardial effusion, or coronary artery abnormalities). A total of 120 patients with MIS-C admitted to the Children's Hospital of Krakow between November 1, 2020, and May 5, 2023, were included in the study (74 during original/alpha dominance, 31 delta, and 15 omicron). Patients in the omicron group were found to be younger than those in the alpha and delta groups (37 vs. 75 vs. 80 months, p = 0.03). The frequency of cardiac failure with systolic function impairment or hypotension was diagnosed more frequently in the original/alpha and delta groups than in the omicron group (44.59% vs. 41.94% vs. 13.33%, p = 0.08) also echocardiographic abnormalities changed, with rates of 60.8%, 35.5%, and 13.3% (p < 0.001) accordingly. The multivariable regression revealed an older age (OR = 1.19, 95% CI = 1.07-1.33, p = 0.002) as the only independent factors of cardiac failure with systolic function impairment or hypotension. In all patients, signs of cardiac failure resolved during the hospitalization. Moreover, in 98.3% of patients, all echocardiagraphic abnormalities resolved completely during the observation period.    Conclusion: The cardiac complications of MIS-C appeared to advance less severely in younger children during the Omicron outbreak. In long-term observation, symptoms of cardiac failure resolve completely. Similarly, also echocardiographic abnormalities normalize in the vast majority of patients. What is Known: • Knowledge about the long-term cardiac complications of MIS-C is still evolving and uncertain. • The greatest concern of MIS-C is cardiac complications, including cardiac failure and coronary artery dilatation. What is New: • Long-term observations revealed complete resolution of cardiac complications in the vast majority of patients with MIS-C, irrespective of the dominant variant. • Cardiac complications of MIS-C were less common in younger children during subsequent pandemic waves in our patient population.

Assessment of salivary cortisol concentrations for procedural pain monitoring in newborns.

OBJECTIVES: The study aimed to evaluate the usefulness of salivary cortisol (SC) for the assessment of procedural pain intensity in preterm and term newborns. METHODS: Three groups of neonates (term, 370-416 weeks; moderate to late preterm, 320-366; and very preterm, <320) hospitalized in neonatal intensive care unit were assessed for the study. Response to nappy change, lung ultrasound (LUS), and blood sampling was analyzed. The intensity of pain was evaluated using continuous heart rate and blood oxygen saturation (SpO2) monitoring, Neonatal Infant Pain Scale (NIPS), and SC concentrations. Saliva samples were collected before and 20 min after the procedure's end. RESULTS: Seventy-one infants were examined: 30 term, 21 moderate to late preterm, and 20 very preterm. SC has increased significantly in response to nappy change only in very preterm newborns (2.13 ng/mL [1.55-3.68] vs. 2.84 ng/mL [1.93-9.06], p = 0.01). LUS did not affect concentrations of SC in any group. Significant increase in SC was observed after blood sampling in term and very preterm infants (2.2 ng/mL [1.45-2.92] vs. 4.29 ng/mL [3.88-5.73], p = 0.002, and 1.88 ng/mL [1.47-4.13] vs. 5.3 ng/mL [3.42-8.02], p = 0.002, respectively). A significant correlation between values of SC increase and NIPS scores was found (Spearman's rank correlation coefficient [rs] = 0.31, p = 0.001). CONCLUSIONS: We observed the increase in SC concentrations in response to painful stimulus. The presence of a correlation between NIPS scores and SC increase suggests that SC can be used as an objective parameter to assess pain in neonates.

Global 5mC and 5hmC DNA Levels in Human Sperm Subpopulations with Differentially Protaminated Chromatin in Normo- and Oligoasthenozoospermic Males.

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

How much, if anything, do we know about sperm chromosomes of Robertsonian translocation carriers?

In men with oligozoospermia, Robertsonian translocations (RobTs) are the most common type of autosomal aberrations. The most commonly occurring types are rob(13;14) and rob(14;21), and other types of RobTs are described as 'rare' cases. Based on molecular research, all RobTs can be broadly classified into Class 1 and Class 2. Class 1 translocations produce the same breakpoints within their RobT type, but Class 2 translocations are predicted to form during meiosis or mitosis through a variety of mechanisms, resulting in variation in the breakpoint locations. This review seeks to analyse the available data addressing the question of whether the molecular classification of RobTs into Classes 1 and 2 and/or the type of DD/GG/DG symmetry of the involved chromosomes is reflected in the efficiency of spermatogenesis. The lowest frequency value calculated for the rate of alternate segregants was found for rob(13;15) carriers (Class 2, symmetry DD) and the highest for rob(13;21) carriers (Class 2, DG symmetry). The aneuploidy values for the rare RobT (Class 2) and common rob(14;21) (Class 1) groups together exhibited similarities while differing from those for the common rob(13;14) (Class 1) group. Considering the division of RobT carriers into those with normozoospermia and those with oligoasthenozoospermia, it was found that the number of carriers with elevated levels of aneuploidy was unexpectedly quite similar and high (approx. 70%) in the two subgroups. The reason(s) that the same RobT does not always show a similar destructive effect on fertility was also pointed out.

Familial Infertility (Azoospermia and Cryptozoospermia) in Two Brothers-Carriers of t(1;7) Complex Chromosomal Rearrangement (CCR): Molecular Cytogenetic Analysis.

Structural aberrations involving more than two breakpoints on two or more chromosomes are known as complex chromosomal rearrangements (CCRs). They can reduce fertility through gametogenesis arrest developed due to disrupted chromosomal pairing in the pachytene stage. We present a familial case of two infertile brothers (with azoospermia and cryptozoospermia) and their mother, carriers of an exceptional type of CCR involving chromosomes 1 and 7 and three breakpoints. The aim was to identify whether meiotic disruption was caused by CCR and/or genomic mutations. Additionally, we performed a literature survey for male CCR carriers with reproductive failures. The characterization of the CCR chromosomes and potential genomic aberrations was performed using: G-banding using trypsin and Giemsa staining (GTG banding), fluorescent in situ hybridization (FISH) (including multicolor FISH (mFISH) and bacterial artificial chromosome (BAC)-FISH), and genome-wide array comparative genomic hybridization (aCGH). The CCR description was established as: der(1)(1qter->1q42.3::1p21->1q42.3::7p14.3->7pter), der(7)(1pter->1p2 1::7p14.3->7qter). aCGH revealed three rare genes variants: ASMT, GARNL3, and SESTD1, which were ruled out due to unlikely biological functions. The aCGH analysis of three breakpoint CCR regions did not reveal copy number variations (CNVs) with biologically plausible genes. Synaptonemal complex evaluation (brother-1; spermatocytes II/oligobiopsy; the silver staining technique) showed incomplete conjugation of the chromosomes. Associations between CCR and the sex chromosomes (by FISH) were not found. A meiotic segregation pattern (brother-2; ejaculated spermatozoa; FISH) revealed 29.21% genetically normal/balanced spermatozoa. The aCGH analysis could not detect smaller intergenic CNVs of few kb or smaller (indels of single exons or few nucleotides). Since chromosomal aberrations frequently do not affect the phenotype of the carrier, in contrast to the negative influence on spermatogenesis, there is an obvious need for genomic sequencing to investigate the point mutations that may be responsible for the differences between the azoospermic and cryptozoospermic phenotypes observed in a family. Progeny from the same parents provide a unique opportunity to discover a novel genomic background of male infertility.

Exhaled carbon monoxide levels correlate with incidence of oral mucosal lesions independent of smoking status.

Carbon monoxide (CO) is an important component of tobacco smoke, but also environmental toxicity. This study analyzed possible correlation between exhaled CO level and oral health indicators in two geographically distributed populations during health-promoting public events organized for local communities in cities with high and low environmental pollution in Poland (907 patients). Self-reported, instructor-led, oral health questionnaire was developed to monitor current and previous mucosal lesion incidence. Exhaled CO correlated with subjects smoking status and environmental CO exposure: highest in smoking inhabitants of Krakow (12 ppm), with lower levels in smokers from Kozienice (6.5 ppm) and non-smokers from Krakow (6 ppm), and lowest for Kozienice non-smokers (2 ppm) (p < 0.001). After propensity score matching and adjustment for smoking status, demography and comorbidities odds ratio for mucosal lesion incidence was 1.46 (1.31-1.63), p < 0.001) per 1 ppm increase of exhaled CO level. This result might implicate a possible role of environmental pollution factors in oral health pathology.

Impact of pre-operative glycated haemoglobin A1C level on 1-year outcomes of endovascular treatment in patients with critical limb ischemia in the course of diabetes mellitus.

INTRODUCTION: Peripheral arterial occlusive disease (PAOD) is a disease with worldwide increasing occurrence. Diabetic patients are greatly exposed on the risk of PAOD and its complications. The aim of the study was to check the influence of preoperative HbA1C on the outcomes of patients with diabetes undergoing PAOD related endovascular treatment. MATERIAL AND METHODS: The study was conducted among 59 patients with PAOD referred from the diabetic foot outpatient for endovascular treatment. They were included in one-year observation based on follow-up visits in 1, 3, 6 and 12 months a er angioplasty and divided into 2 groups basing on their preoperative glycaemia. The clinical condition of the lower limbs was assessed by use of the Rutherford classification, ankle-brachial index (ABI) and toe-brachial index (TBI). Changes in patients' quality of life (QoL) were also evaluated. RESULTS: Reintervention within 12 months were less frequent in patients with HbA1C ≤8.0% than in HbA1C >8.0% patients (9.09% vs. 35.48%, p = 0.03). TBI of the treated limb was lower in patients with elevated than in patients with proper glycaemia at 6 month [0.2 (0.0-0.38) vs. 0.38 (0.31-0.46); p <0.008] and 12 month follow-up [0.17 (0.0-0.27) vs. 0.32 (0.25-0.38); p <0,001]. The rate of healed ulcerations after 6 months was higher in patients HbA1C ≤8.0% (45.0% vs. 16.13%; p = 0.02) and they had significantly greater improvement of QoL. CONCLUSION: Results of this study shows that preoperative level of glycaemia is an important factor for long-term prognosis in diabetic patients with PAOD. Elevated HbA1C level decreases significantly long-term improvement of QoL in DM patients undergoing endovascular treatment.

X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.

BACKGROUND: The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS: We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS: We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS: In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

Coronary Artery Disease in Young Adults: Who Needs Surgical Revascularization? A Retrospective Cohort Study.

BACKGROUND: Coronary artery disease (CAD) is a major cause of death and disability in developed countries. Despite the fact that prevalence accrues with age, an increasing number of young patients suffering from CAD is being observed worldwide. The aim of this study is to describe the population of young adults suffering from CAD and requiring coronary artery bypass grafting (CABG), and to assess early outcomes after the procedure. METHODS: A retrospective cohort study analyzed 190 consecutive patients aged ≤50 years old that underwent CABG between 2010 and 2014. Baseline characteristics and operative data were presented in the study. Postoperative complications, such as major adverse cardiac and cardiovascular events (MACCE), prolonged mechanical ventilation (>72 hours), bleeding requiring reexploration, sternal dehiscence, and others were assessed. RESULTS: A population comprising mostly overweight or obese males with a mean age of 46 ± 4.1 years was analyzed. Patients suffered mostly from three-vessel disease (81%), hypertension (74.7%), and had previous history of myocardial infarction (MI) (60%). The majority of patients had normal left ventricle ejection fraction (LVEF) (83.1%). 22.6% of cases were emergent procedures. Perioperative mortality was low (1%) and overall MACCE rate stood at 2.6%. Emergent surgery was associated with a higher incidence of postoperative complications (P = .007). The number of diseased vessels, LVEF, and CCS/NYHA class-on-admission was not associated with a higher incidence of postoperative complications (P > .05 for all). CONCLUSION: CAD in young patients remains an issue described insufficiently in the literature. Among our study cohort of younger patients undergoing CABG, the majority of the patients had multivessel disease and were slightly symptomatic with normal LVEF. Although the postoperative complication rate was low, the percentage of emergent surgeries was alarmingly high in this population. Consistent with the literature, we highlight the importance of CAD screening in the young population to detect subclinical disease, which might be treated therapeutically or operated electively.

In diabetic Charcot neuroarthropathy impaired microvascular function is related to long lasting metabolic control and low grade inflammatory process.

AIMS: The aim of this study was to assess microvascular function associated with the occurrence of Charcot neuroarthropathy (CN) in patients with diabetes. METHODS: We evaluated 70 diabetic patients (54 men) with Charcot neuroarthropathy (CN-DM), median age 59 (IQR: 51-62), mean disease duration 16±8years. The control group were 70 subjects with diabetes and without Charcot neuroarthropathy (DM), 54 men, median age 60 (54-62), mean diabetes duration 15±7years. We assessed metabolic control of diabetes, serum C-reactive protein concentration (CRP) and cardiovascular autonomic neuropathy (CAN). We used AGE-Reader to measure skin autofluorescence (AF) associated with accumulation of advanced glycation end products that reflects long lasting metabolic control. Microvascular function was examined by laser Doppler flowmetry (PERIFLUX 5000) with thermal hyperemia (TH) and postocclusive reactive hyperemia (PORH). RESULTS: CN-DM patients as compared to DM subjects had lower HbA1c level [7.6 (6.6-8.4) vs 8.4 (7.3-9.7)%, p<0.001], lower eGFR [75.9±24.1 vs 86.6±17.8ml/min/1.73m(2), p=0.003], higher CRP serum concentration [3.8 (2.3-10.1) vs 1.9 (0.8-4.4)mg/l, p<0.001] and higher skin autofluorescence [2.8 (2.5-3.1) vs 2.6 (2.3-2.9)AU, p=0.03]. The cardiovascular autonomic neuropathy (CAN) was more frequently diagnosed in CN-DM subjects [59 vs 27%, p<0.001]. The peak flow during thermal hyperemia (THmax) was lower in CN-DM subjects as compared to DM group [156 (93-240) vs 235 (155-300)PU, p=0.001]. We found negative correlation between THmax and CRP concentration (Rs=-0.34, p=0.003), TG concentration (Rs=-0.37, p=0.002) and skin AF (Rs=-0.32, p=0.04) and positive correlation between THmax and HDL cholesterol level (Rs=0.42, p<0.001) in CN-DM patients. There was also a positive correlation between PORHpeak and HDL cholesterol level (Rs=-0.23, p=0.04). CONCLUSION: Deterioration of microvascular function and autonomic system dysfunction are present in Charcot neuroarthropathy. Impaired microvascular reactivity is associated with worse long lasting metabolic control of diabetes and low grade inflammatory process.

FISH and array CGH characterization of de novo derivative Y chromosome (Yq duplication and partial Yp deletion) in an azoospermic male.

This study presents a 28-year-old infertile male who was referred to the cytogenetic laboratory for chromosomal analysis after 4 years of regular unprotected intercourse in whom non-obstructive azoospermia was revealed. Standard cytogenetic G-banding was performed on metaphase spreads and a de-novo karyotype 46,X,der(Y)(q11.22;p11.3) was identified. This analysis was followed by flourescence in-situ hybridization(FISH) and array comparative genomic hybridization (aCGH). Finally, the patient's karyotype was identified as 46,X,der(Y)(qter→q11.221::p11.31→qter).ish der(Y) (qter+,pter-,SHOX+,SRY+,Ycen+,DYZ3+;DYZ1+,qter+).arrYq11.221q12(14,448,863-59,288,511) x2, Yp11.32p11.31(104,062-266,388) x0. It is proposed that de-novo derivative monocentric Y chromosome with duplicated region Y qter→q11.221::p11.31→qter with partial deletion of Yp PAR1 region most probably can perturb the conjugation of sex chromosomes during first meiotic division of spermatogenic arrested differentiation (development).

A case report of severe panhypopituitarism in a newborn delivered by a women with Turner syndrome.

Turner syndrome (TS) is a congenital disease caused by absence or structural abnormalities of sex chromosomes resulting in gonadal dysgenesis. Spontaneous pregnancies occur in 2-8% of patients, especially with mosaic kariotypes, however they are associated with increased risk of poor outcome both for mother and fetus. We report a 4-day-old male infant delivered by women with mosaic TS who was admitted to the pediatric intensive care unit and presented with severe panhypopituitarism as the early manifestation of pituitary stalk interruption syndrome (PSIS). To the best of our knowledge this is the first report of severe panhypopituitarism in a newborn borne by women with TS.

Recurrence risks for different pregnancy outcomes and meiotic segregation analysis of spermatozoa in carriers of t(1;11)(p36.22;q12.2).

Cumulative data obtained from two relatively large pedigrees of a unique reciprocal chromosomal translocation (RCT) t(1;11)(p36.22;q12.2) ascertained by three miscarriages (pedigree 1) and the birth of newborn with hydrocephalus and myelomeningocele (pedigree 2) were used to estimate recurrence risks for different pregnancy outcomes. Submicroscopic molecular characterization by fluorescent in situ hybridization (FISH) of RCT break points in representative carriers showed similar rearrangements in both families. Meiotic segregation patterns after sperm analysis by three-color FISH of one male carrier showed all possible outcomes resulting from 2:2 and 3:1 segregations. On the basis of empirical survival data, we suggest that only one form of chromosome imbalance resulting in monosomy 1p36.22→pter with trisomy 11q12.2→qter may be observed in progeny at birth. Segregation analysis of these pedigrees was performed by the indirect method of Stengel-Rutkowski and showed that probability rate for malformed child at birth due to an unbalanced karyotype was 3/48 (6.2±3.5%) after ascertainment correction. The risk for stillbirths/early neonatal deaths was -/48 (<1.1%) and for miscarriages was 17/48 (35.4±6.9%). However, the probability rate for children with a normal phenotype at birth was 28/48 (58.3±7.1%). The results obtained from this study may be used to determine the risks for the various pregnancy outcomes for carriers of t(1;11)(p36.22;q12.2) and can be used for genetic counseling of carriers of this rearrangement.

Sperm FISH and chromatin integrity in spermatozoa from a t(6;10;11) carrier.

Complex chromosome rearrangements (CCRs) are structurally balanced or unbalanced aberrations involving more than two breakpoints on two or more chromosomes. CCRs can be a potential reason for genomic imbalance in gametes, which leads to a drastic reduction in fertility. In this study, the meiotic segregation pattern, aneuploidy of seven chromosomes uninvolved in the CCR and chromatin integrity were analysed in the ejaculated spermatozoa of a 46,XY,t(6;10;11)(q25.1;q24.3;q23.1)mat carrier with asthenozoospermia and a lack of conception. The frequency of genetically unbalanced spermatozoa was 78.8% with a prevalence of 4:2 segregants of 38.2%, while the prevalence of the adjacent 3:3 mode was 35.3%. Analysis of the aneuploidy of chromosomes 13, 15, 18, 21, 22, X and Y revealed an approximately fivefold increased level in comparison with that of the control group, indicating the presence of an interchromosomal effect. Sperm chromatin integrity status was evaluated using chromomycin A3 and aniline blue staining (deprotamination), acridine orange test and TUNEL assay (sperm DNA fragmentation). No differences were found when comparisons were made with a control group. We suggest that the accumulation of genetically unbalanced spermatozoa, significantly increased sperm aneuploidy level and decreased sperm motility (20%, progressive) were not responsible for the observed lack of reproductive success in the analysed infertile t(6;10;11) carrier. Interestingly, in the case described herein, a high level of sperm chromosomal imbalance appears not to be linked to sperm chromatin integrity status.

Effects of Tcte1 knockout on energy chain transportation and spermatogenesis: implications for male infertility.

STUDY QUESTION: Is the Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1-DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations. STUDY DESIGN SIZE DURATION: Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS SETTING METHODS: To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous Tcte1+/- and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein-protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In Tcte1-/- males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme. LARGE SCALE DATA: All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request. LIMITATIONS REASONS FOR CAUTION: In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked. WIDER IMPLICATIONS OF THE FINDINGS: This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the 'male infertility list' because of its crucial role in spermatogenesis and proper sperm functioning. STUDY FUNDING/COMPETING INTERESTS: This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

The influence of repeated pain exposure on morning salivary cortisol in term and preterm neonates.

INTRODUCTION: Introduction: Because neonates in the intensive care units (ICU) experience recurrent stress due to painful medical procedures, they are at risk of dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis. Aim of the study: To evaluate the influence of repeated pain exposure on morning salivary cortisol (SC) in newborns admitted to the ICU. MATERIAL AND METHODS: The neonates were divided into 3 groups: term (370/7-416/7 weeks), moderate to late preterm (320/7-366/7 weeks), and very preterm (< 320/7 weeks). The hospital stay was prospectively monitored for the number of the most common medical procedures. At least 2 saliva samples for morning SC were collected after completion of 35 weeks of postmenstrual age (PMA) in preterm infants and before discharge in term neonates. The results of SC were compared with the reference intervals for healthy term newborns. RESULTS: The study group consisted of 57 patients: 21 term, 17 moderate to late preterm, and 19 very preterm neonates. Very preterm neonates obtained the highest values of mean morning SC in comparison to moderate to late preterm and term infants (3.83 [1.67-8.81] ng/ml vs. 2.44 [1.94-4.38] ng/ml vs. 2.15 [1.5-5.25] ng/ml, p = 0.45). The relationship between mean morning SC and the number of invasive blood samplings was found only in term newborns (Rs = -0.44, p < 0.05). 46% of all SC measurements in very preterm, 47% in moderate to late preterm, and 46% in term infants were within the reference intervals for healthy newborns. CONCLUSIONS: High exposure to painful procedures seems to dampen the morning SC in term, but not in preterm infants.

Epigenetic markers in the embryonal germ cell development and spermatogenesis.

Spermatogenesis is the process of generation of male reproductive cells from spermatogonial stem cells in the seminiferous epithelium of the testis. During spermatogenesis, key spermatogenic events such as stem cell self-renewal and commitment to meiosis, meiotic recombination, meiotic sex chromosome inactivation, followed by cellular and chromatin remodeling of elongating spermatids occur, leading to sperm cell production. All the mentioned events are at least partially controlled by the epigenetic modifications of DNA and histones. Additionally, during embryonal development in primordial germ cells, global epigenetic reprogramming of DNA occurs. In this review, we summarized the most important epigenetic modifications in the particular stages of germ cell development, in DNA and histone proteins, starting from primordial germ cells, during embryonal development, and ending with histone-to-protamine transition during spermiogenesis.

RéSUMé: La spermatogenèse est le processus de génération de cellules reproductrices mâles à partir de cellules souches spermatogoniales, dans l'épithélium séminifère du testicule. Au cours de la spermatogenèse, des événements spermatogéniques clés tels que l'auto-renouvellement des cellules souches et l'engagement dans la méiose, la recombinaison méiotique, l'inactivation méiotique du chromosome sexuel, suivis d'un remodelage cellulaire et chromatique des spermatides allongées se produisent, conduisant à la production de spermatozoïdes. Tous les événements mentionnés sont au moins partiellement contrôlés par les modifications épigénétiques de l'ADN et des histones. De plus, au cours du développement embryonnaire, une reprogrammation épigénétique globale de l'ADN se produit dans les cellules germinales primordiales. Dans cette revue, nous avons résumé les modifications épigénétiques les plus importantes dans les étapes particulières du développement des cellules germinales, dans l'ADN et les protéines histones, en partant des cellules germinales primordiales, au cours du développement embryonnaire, jusqu'à la transition histone-protamine pendant la spermiogenèse.

Proteomic approach towards identification of seminal fluid biomarkers from individuals with severe oligozoospermia, cryptozoospermia and non-obstructive azoospermia: a pilot study.

Background: Infertility becomes a global problem that affects to the same extent females and males. As reasons of male infertility can differ among individuals, the accurate diagnostics is essential for effective treatment. The most problematic both in diagnostics and in treatment are disturbances of spermatogenesis. Seminal fluid is rich in proteins that potentially can serve as markers for male infertility and among them, markers of spermatogenesis which are highly desired. Methods: To find biomarkers of spermatogenesis, we applied comparative proteomics using nano ultra performance liquid chromatography and tandem mass spectrometry (nanoUPLC-MS/MS) followed by single-sample Western blotting (WB) using seminal fluid samples from males with different types of infertility including non-obstructive azoospermia (NOA), cryptozoospermia (C) and severe oligozoospermia (SO). Then, the extensive survey on the identified proteins and their function in male reproductive system has been done. Results: The proteomic approach has enabled to identified five seminal fluid proteins being potential markers of spermatogenesis disorders: ADGRG2, RAB3B, LTF, SLC2A3 and spermine synthase (SMS). Among them ADGRG2 seems to be strongly involved in male infertility. In addition, WB indicated that the distribution of LTF, SLC2A3 and SMS was not coherent among the individuals, especially in a group with NOA. Functional annotation analysis and search in proteomics databases revealed that vast majority of the proteins originated from extracellular environment. Conclusions: The presented data point out several proteins that potentially can become biomarkers of male infertility. The data suggest, however, different mechanisms behind the male infertility indicating that the etiology is more complex. We assume that recognition of these mechanisms may lead to the creation of specific protein panel helpful in the management of male infertility and therefore, further studies are required.